| The helper component-proteinase (HC-Pro) encoded by potyviruses is a multifunctionalprotein involved in many processes in life cycle, including genome amplification, movement,symptom expression, aphid transmission, RNA silencing suppression and synergism withviruses of other genus. In the present study, we analyzed the effects of different mutants ofpotato virus A (PVA) and tobacco vein banding mosaic virus (TVBMV) HC-Pro on theactivities of RNA silencing suppression (RSS), synergism, and virulence. The main resultsread as follows.We first constructed a transient assay system for RNA silencing suppression thatincludes three vectors, pCaGFP, pCahpGFP and pCaHC (carrying TVBMV HC-Pro gene).With this system, we determined the activities of wild type and18mutants on RSS. The RSSactivities of TVBMV HC-Pro mutants were classified into four categories:(1) Five mutantsshowed increased activity;(2) Five mutants showed similar RSS activity with wild typeHC-Pro;(3) Two showed reduced activity;(4) Six lost RSS activity completely. More GFPwas detected in Nicotiana benthamiana leaf patches agroinfiltrated TVBMV HC-Pro mutantsthat showed stronger RSS activity. There is no GFP detected in N. benthamiana leaf patchesagoinfiltrated TVBMV HC-Pro mutants that lost their RSS activity.The RSS activities of wild type and18mutants of PVA HC-Pro were compared usingthree vectors, pAGFP, pAhpGFP and pAHC (carrying PVA HC-Pro gene). The resultsshowed that nine mutants showed reduced RSS activities, and the other nine mutants losttheir RSS activities. Same mutation may show different effects on RSS activity in differentviruses. For examples, the mutation D198A partially reduced the RSS activity of TVBMVHC-Pro, but abolished that of PVA HC-Pro; F6Del reduced the RSS activity of PVA HC-Pro,but abolished that of TVBMV HC-Pro.Recombinant PVX vectors carrying TVBMV and PVA HC-Pro gene will result in systemic necrosis of N. benthamiana plants. The same mutations of TVBMV and PVAHC-Pro gene were analyzed for their synergism activity. Twelve mutants of TVBMV HC-Proresulted in systemic necrosis as the wild type one, while other six mutants just inducedmosaic. The mutations in TVBMV HC-Pro gene that abolished RSS activity also lost thesynergism activity, indicating the association between these two functions. Eleven PVAHC-Pro mutants lost their synergism activity, among which nine lost their RSS activity andtwo showed reduced RSS activity.The effects of17TVBMV HC-Pro mutants on the virulence of TVBMV were analyzedwith pCaTVBMV-GFP, an infectious clone carrying gfp gene. As wild type pCaTVBMV-GFP,mutants EITC, AGS and SQN induced vein clearing, mosaic and malformation in N.benthamiana plants, implying that these mutations had no effect on virulence of TVBMV.Mutants APS, PAK, D198Y, D198F and D198G showed slight vein clearing8dpi and similarsymptom as wild type virus later, implying that these mutations just delayed the symptomexpression. Mutants D198H, D198R, D198K, FINK, F6Del, D198A, DEN, IQI and ARTinduced slight symptom on N. benthamiana plants, from which only weak fluorescence wasobserved. The mutants that showed abolished RSS activity also showed reduced virulence,imply these two processes were closely related.
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